Abstract Background: New approaches to therapeutic development using cellular engineering are rapidly emerging and evolving. Applications such as, but not limited to, chimeric antigen receptor (CAR)-T cells, regenerative medicine, and drug delivery using liposomes and nanoparticles are steadily transitioning from idea to reality. In fact, such cellular therapeutics are increasingly becoming a viable option for treatment of patients with cancer and genetic diseases, with the first two CAR-T therapies receiving FDA approval in 2017. However, it is becoming increasingly clear with CAR-T that multiple editing steps will be needed to confer the desired phenotype, as well as improved safety and toxicity profiles. Current approaches for gene editing and intracellular delivery include chemical methods (e.g., lipofectaime), electroporation, and viral vectors. Chemical methods are widely used in research labs because they are affordable and easy to use, but they are not very effective at transfecting primary cells. Electroporation is another widely used method that is capable of treating primary cells, but it is only effective at delivering highly charged molecules, i.e. nucleic acids. Viral vectors were the earliest gene delivery technology, they are quite efficient at delivering DNA, but are also limited to nucleic acid delivery and have safety concerns related to viral integration in the genome. With the growing interest in cellular engineering, a need for more efficient and flexible intracellular delivery technologies has emerged. OpenCell Technologies (OCT) has developed a proprietary technology, POROS, to deliver macromolecules such as DNA, RNA, protein, liposomes and nanoparticles to a wide variety of cell types. POROS uses acoustic waves to drive cells through an array of nozzles one cell at a time, thus creating a mechanoshear force to porate cells in a uniform manner. The acoustic shear poration (ASP) is coupled with an electrophoresis (EP) step that uses a low strength electric field to actively drive molecules into the cells through the pores already created. In this SBIR project, OCT will expand the capabilities of its POROS platform by developing the POROS-multipayload delivery (MPD) platform. Approach: In this Phase I feasibility study, OCT will demonstrate that its delivery technology, POROS, can be combined with unique reagents and protocols to enable efficient delivery of multiple large molecules to cells. In this project, we will first optimize protocols for nucleic acid, protein, liposome and nanoparticle delivery using three model systems that use reporter gene readouts, such as GFP. Following protocol development and optimization, this technology will be tested for two relevant applications, lentivirus packaging for viral gene delivery and CAR-T cell production using CRISPR/Cas9. Both of these applications require delivery of two or more large molecular payloads and will serve as the proof-of-concept that the POROS-MPD platform improves delivery.